One of the most promising methodologies in gene delivery is the use of adenovirus and adeno-associated virus as a viral vector. One of the major challenges in using adenovirus to deliver DNA to cells is that it is very difficult to create viruses carrying the DNA that also lack the viral E4 region. Adenovirus vectors that lack the E4 region are desirable because they can accept larger inserts of DNA and because they cannot express a toxic and mutagenic protein, the E4orf6 protein. It is difficult to create these viruses and to produce sufficient amounts of these viruses because the viral E4orf6 protein is needed for replication, but is cytotoxic to the cells in which the virus is replicated when supplied in trans. This cytopathic effect of the E4orf6 protein has been a severe roadblock to the development of successful nucleic acid delivery systems that use adenovirus and/or AAV.
Open reading frame 6 of the early region 4 (E4orf6) of group C adenovirus (Ad) encodes a multifunctional protein that enhances viral replication (reviewed in Leppard (1997) J. Gen. Virol. 78:2131-2138) and acts as an oncoprotein (Nevels et al. (1997) Proc. Natl. Acad. Sci. USA 94:1206-1211). Ad mutants that lack the entire E4 region are severely defective for viral DNA replication and late viral protein synthesis (Bridge and Ketner (1990) Virology 174:345-353; Halbert et al. (1999) J. Virol. 56:250-257; Huang and Hearing (1989) J. Virol. 63:2605-2615; Weiden and Ginsberg (1994)Proc. Natl. Acad. Sci. USA 91:153-157). However, expression of the E4orf6 protein in trans largely corrects the growth defect of an E4-deletion virus (Armentano et al. (1995) Hum. Gene Ther. 6:1343-1353; Halbert et al. (1985) J. Virol. 56:250-257; Ketner et al. (1989) Nucleic Acids Res. 17:3037-3048).
The E4orf6 protein increases late viral protein production by facilitating the cytoplasmic accumulation of mRNA at a postranscriptional level (Leppard (1997) J. Gen. Virol. 78:2131-2138; Nordqvist et al. (1994) Mol. Cell Biol. 14:437-445; Pilder et al. (1986) Mol. Cell Biol. 6:470-476). In addition to enhancing the processing and stability of late viral RNA in the nucleus (Dix and Leppard (1993) J. Virol. 67:3226-3231; Ohman et al. (1993) Virology 194:50-58), the E4orf6 protein, as part of a complex with the E1B-55 kDa protein, promotes the nucleocytoplasmic transport of processed late viral mRNA (Pilder et al. (1986) Mol. Cell Biol. 6:470-476; Rubenwolf et al. (1997) J. Virol. 71:1115-1123; Sarnow et al. (1984) J. Virol. 49:692-700). Additionally, the E4orf6-E1B-55 kDa protein complex blocks the nucleocytoplasmic transport of most host mRNAs (Babiss et al. (1985) Mol. Cell Biol. 5:2552-2558; Pilder et al. (1986) Mol. Cell Biol. 6:470-476).
It has been proposed that the E4orf6-E1B-55 kDa protein complex binds a key component of the host cell nucleocytoplasmic transport system to achieve the selective transport of late viral mRNA (Ornelles and Shenk (1991) J. Virol. 65:424-429). The E4orf6 protein also interferes with the host cell cycle, and in cooperation with the E1proteins of Ad, promotes oncogenesis of baby rat kidney (BRK) cells (Nevels et al. (1997) Proc. Natl. Acad. Sci. USA 94:1206-1211; Nevels et al. (1999) Oncogene 18:9-17). The E4orf6-mediated transformation of BRK cells may stem from the ability to bind and inactivate tumor suppressor proteins such as p53 or p73 (Dobner et al. (1996) Science 272:1470-1473; Higashino et al. (1998) Proc. Natl. Acad. Sci USA 95:15683-15687; Steegenga et al. (1999) Mol. Cell Biol. 19:3885-3894), to bind and inactivate the cyclin A protein (Grifman et al. (1999) J. Virol. 73:10010-10019), or to increase the host cell mutation rate (Moore et al. (1996) Proc. Natl. Acad. Sci. USA 93:11295-11301).
Some of the critical features of the E4orf6 protein required for its function have been identified. A protein fragment containing the amino-terminal 58 amino acids of the E4orf6 protein binds both the E1B-55 kDa protein and the tumor suppressor p53 protein in vitro (Dobner et al. (1996) Science 272:1470-1473; Rubenwolf et al. (1997) J. Virol. 71:1115-1123). Although the E4orf6/7 protein contains this sequence of amino acids and binds the E1B-55 kDa and p53 proteins in vitro, it cannot establish a functional interaction with the E1B-55 kDa protein in the cell (Orlando and Ornelles (1999) J. Virol. 73:4600-4610), elicit p53 degradation (Querido et al. (1997) J. Virol. 71:788-798), or induce transformation of BRK cells (Nevels et al. (1997) Proc. Natl. Acad. Sci. USA 94:1206-1211).
It has been reported that the E4orf6 protein contains a cryptic leucine-rich nuclear export signal (NES), centered around isoleucine-90 and leucine-92 (Dobbelstein et al. (1997) EMBO J. 16:4276-4284) that protein is necessary for E4orf6-mediated degradation of p53 (Nevels et al. (2000) J. Virol. 74:5168-5181). Although E4orf6 proteins lacking the amino terminus or the NES can cooperate with the E1B and E1A proteins to transform BRK cells, these cells are not as tumorogenic in nude mice cells as BRK cells transformed with the wild-type E4orf6 protein (Nevels et al. (2000) J. Virol. 74:5168-5181).
Several cysteine and histidine residues that are conserved between E4orf6 proteins from several Ad subgroups are essential for many functions of the E4orf6 protein. E4orf6 variants with substitutions among these amino acids fail to promote late viral gene expression, no longer co-immunoprecipitate with the E1B-55 kDa protein, fail to direct nuclear localization of the E1B-55 kDa protein, fail to promote destabilization of the p53 protein, transform BRK cells with reduced efficiency, and produce transformed cells with diminished oncogenic potential in nude mice (Boyer and Ketner (2000) J. Biol. Chem. 275:14969-14978; Nevels et al. (2000) J. Virol. 74:5168-5181). Boyer and Ketner have suggested that these conserved cysteine and histidine residues coordinate with two or more zinc ions to establish the proper tertiary structure of the E4orf6 protein (Boyer and Ketner (2000) J. Biol. Chem. 275:14969-14978).
The arginine-faced amphipathic α helix at the carboxy terminus of the E4orf6 protein is required for many of the functions of the E4orf6 protein. E4orf6 variants that lack this structure or contain proline substitutions within the α helix fail to promote virus replication (Orlando and Ornelles (1999) J. Virol. 73:4600-4610). Additionally, these E4orf6 variants fail to relocalize the E1B-55 kDa protein to the nucleus of cotransfected cells. The integrity of the arginine-faced amphipathic α helix is also required for E4orf6mediated p53 degradation (Nevels et al. (2000) J. Virol. 74:5168-5181). Furthermore, it has been suggested that destabilization of p53 by the E4orf6 protein depends on binding both the E1B-55 kDa protein as well as uncharacterized cellular factors (Querido et al. (2001) J. Virol. 75:699-709). An intact arginine-faced amphipathic α helix is also required for the full oncogenic potential of the E4orf6 protein as measured by the ability to transform BRK cells and elicit an abnormal state of growth termed hypertransformation (Nevels et al. (2000) J. Virol. 74:5168-5181). Although these oncogenic functions may depend in part, on the E1B-55 kDa protein, it is possible that the arginine-faced amphipathic α helix of the E4orf6 protein interacts with some cellular factors that control cell growth. For example, a motif within the amphipathic α helix was suggested to bind cyclin A and augment expression of a transgene present on a recombinant adeno-associated virus (rAAV) (Grifman et al. (1999) J. Virol. 73:10010-10019). Since the E4orf6 effect on rAAV transgene expression resembled that seen upon treatment of rAAV-infected cells with inhibitors of DNA synthesis or DNA damaging agents, it is possible that the E4orf6 protein can perturb either the integrity of cellular DNA or the signaling pathways associated with DNA damage (Alexander et al. (1994) J. Virol. 68:8282-8287; Ferrari et al. (1996) Science 272:1470-1473; Jansen-Durr (1996) Trends Genet. 12:270-275).
As indicated above, it would be extremely useful to provide a way to supply the necessary functions of the E4orf6 protein in a helper cell for the production of viral vectors, while also reducing the cytotoxic effects of this protein.